The Streptococci make up a medically important genera of microbes known to cause several types of disease in humans, including, for example, otitis media, conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pleural empyema and endocarditis, and most particularly meningitis, such as for example infection of cerebrospinal fluid. Since its isolation more than 100 years ago, Streptococcius pneumoniae has been one of the more intensively studied microbes. For example, much of our early understanding that DNA is, in fact, the genetic material was predicated on the work of Griffith and of Avery, Macleod and McCarty using this microbe. Despite the vast amount of research with S. pneumoniae, many questions concerning the virulence of this microbe remain. It is particularly preferred to employ Streptococcal genes and gene products as targets for the development of antibiotics.
The frequency of Streptococcus pneumoniae infections has risen dramatically in the past 20 years. This has been attributed to the emergence of multiply antibiotic resistant strains and an increasing population of people with weakened immune systems. It is no longer uncommon to isolate Streptococcus pneumoniae strains which are resistant to some or all of the standard antibiotics. This has created a demand for both new anti-microbial agents and diagnostic tests for this organism.
The enzyme UDP-N-acetylmuramoylalanine:D-glutamate ligase, encoded by the gene MurD catalyses the addition of the second amino acid (D-glutamate) of the peptide moiety in peptidoglycan biosynthesis. D-glutamate is added to UDP-N-acetyl muramoyl-L-alanine with the concomittant hydrolysis of ATP to yield UDP-N-acetylmuramoyl-L-alanine-D-glutamate, ADP and pyrophosphate. The gene has been cloned and sequenced from Escherichia coli and the corresponding protein has been over-expressed, purified and kinetically characterised (Pratviel-Sosa F, Mengin-Lecreulx D & van Heijenoort J (1991) Eur. J. Biochem, 202, 1169-1176). The crystal structure has also been solved (Betrand, J. A, Auger, G., Fanchon, E., Martin, L., Blanot, D., van Heijenoort, J. & Dideberg, O., (1997) EMBO J., 16, 3416-3425). MurD has also been sequenced from such organisms as Bacillus subtilis, Haemophilus influenzae and Enterococcus faecalis.
The discovery of a MurD homologue in the human pathogen Streptococcus pneumoniae will allow us to produce UDP-N-acetylmuramoylalanine:D-glutamate ligase enzyme which can then be used to screen for novel inhibitors. Inhibitors of this protein have utility in anti-bacterial therapy as they will prevent the construction of the bacterial cell wall.
Clearly, there is a need for factors, such as the novel compounds of the invention, that have a present benefit of being useful to screen compounds for antibiotic activity. Such factors are also useful to determine their role in pathogenesis of infection, dysfunction and disease. There is also a need for identification and characterization of such factors and their antagonists and agonists which can play a role in preventing, ameliorating or correcting infections, dysfunctions or diseases.
The polypeptides of the invention have amino acid sequence homology to a known MurD from Enterococcus faecalis protein. See Genembl database, Accession Number U94707; Pratviel-Sosa et al., Eur. J. Biochem, 202, 1169-1176 (1991); Betrand et al., EMBO J., 16, 3416-3425 (1997); and Itaya and Ui, Clin.Chim.Acta 14,361-366 (1966).